This invention relates to the PrtR-PrtK cell surface protein of Porphyromonas gingivalis and in particular a multimeric cell associated protein complex comprising the PrtR and PrtK proteins. The invention also relates to pharmaceutical compositions and associated agents based on said complex for the detection, prevention and treatment of Periodontal disease associated with P. gingivalis. 
Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth""s supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is P. gingivalis as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas P. gingivalis is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of P. gingivalis in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of P. gingivalis. These findings in both animals and humans suggest a major role for P. gingivalis in the development of adult periodontitis.
P. gingivalis is a black-pigmented, anaerobic, asaccharolytic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganism has an absolute growth requirement for iron, preferentially in the form of haeme or its Fe(III) oxidation product haemin and when grown under conditions of excess haemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of P. gingivalis including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. In particular, proteases have received a great deal of attention for their ability to degrade a broad range of host proteins including structural proteins and others involved in defence. The proteins that have been shown to be substrates for P. gingivalis proteolytic activity include collagen types I and IV, fibronectin, fibrinogen, laminin, complement and plasma clotting cascade proteins, xcex11-antitrypsin, xcex12-macroglobulin, antichymotrypsin, antithrombin III, antiplasmin, cystatin C, IgG and IgA. The major proteolytic activities associated with this organism have been defined by substrate specificity and are xe2x80x9ctrypsin-likexe2x80x9d, that is cleavage on the carboxyl side of arginyl and lysyl residues and collagenolytic although other minor activities have been reported.
P. gingivalis trypsin-like proteolytic activity has been shown to degrade complement, generating biologically active C5a, impair the phagocytic and other functions of neutrophils by modifying surface receptors, and abrogate the clotting potential of fibrinogen prolonging plasma clotting time. The trypsin-like proteolytic activity of P. gingivalis also generates Fc fragments from human IgG1 stimulating the release of pro-inflammatory cytokines from mononuclear cells and is associated with vascular disruption and enhanced vascular permeation through the activation of the kallikrein-kinin cascade. P. gingivalis spontaneous mutants with reduced trypsin-like activity as well as wild-type cells treated with the trypsin-like protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone are avirulent in animal models. Further, it has been shown that P. gingivalis grown under controlled, haemin-excess conditions expressed more trypsin-like and less collagenolytic activity and were more virulent in mice relative to cells grown under haemin-limited but otherwise identical conditions. The increased expression of the trypsin-like activity by the more virulent P. gingivalis has led to the speculation that the trypsin-like proteolytic activity may be the major determinant for infection or disease. However, the cell-associated trypsin-like proteolytic activities of P. gingivalis have not been characterised to date.
There has been considerable endeavour to purify and characterise the trypsin-like proteases of P. gingivalis from cell-free culture fluids. Chen et al, (1992) [J Biol Chem 267:18896-18901] have purified and characterised a 50 kDa arginine-specific, thiol protease from the culture fluid of P. gingivalis H66 designated Arg-gingipain. A similar arginine-specific thiol protease has been disclosed in JP 07135973 and the amino acid sequence disclosed in WO 9507286 and in Kirszbaum et al, 1995 [Biochem Biophys Res Comm 207:424-431]. Pike et al (1994) [J Biol Chem 269:406-411] have characterised a 60 kDa lysine-specific cysteine proteinase from the culture fluid of P. gingivalis H66 designated Lys-gingipain and the partial gene sequence for this enzyme was disclosed in WO 9511298 and fully disclosed in WO 9617936. However, prior to the development of the present invention it was unknown that there existed on the cell surface of P. gingivalis a 300 kda complex of arginine-specific and lysine-specific proteases both containing adhesin domains. The 300 kDa complex has been designated the PrtR-PrtK complex. The presence of the PrtR-PrtK cell surface complex exhibiting both arginine- and lysine-specific proteolytic activity together with adhesin activity was previously unknown. Furthermore, the new PrtR-PrtK complex of the present invention is expressed on the cell surface, is a major virulence-associated factor and contains unique epitopes not displayed on the individual domains. The previously disclosed arginine-specific and lysine-specific thiol proteases, as discussed, do not exhibit any of these features and have proven of limited application to date. However, the aforementioned features have rendered the PrtR-PrtK complex of the invention ideal for development of diagnostic and immunoprophylactic products. The PrtR-PrtK cell surface complex is accordingly of particular interest for diagnostics and neutralisation by passive immunity through oral compositions containing neutralising antibodies and by vaccine development. In particular for the development of an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing an inactivated PrtR-PrtK is a genetically engineered commensal inhabitant of the oral cavity.
Accordingly in a first aspect the present invention consists in a substantially purified antigenic complex for use in raising an antibody response directed against Porphyromonas gingivalis, the complex comprising at least one multimeric protein complex of arginine-specific and lysine-specific thiol endopeptidases each containing at least one adhesin domain, the complex having a molecular weight of greater than about 200 kDa.
In the context of this disclosure, the terms xe2x80x9cadhesinxe2x80x9d and xe2x80x9chemagglutininxe2x80x9d may be considered to be synonymous.
In a preferred form of the present invention the multimeric protein complex is associated with virulent strains of Porphyromonas gingivalis, preferably has a molecular weight of about 294 to about 323 kDa and is preferably derived from P. gingivalis W50.
It is also preferred that the multimeric protein complex is composed of 9 proteins. These 9 proteins preferably have the following N-terminal sequences:
DVYTDHGDLYNTPVRML (SEQ ID NO: 1)
YTPVEEKQNGRMIVIVAKKYEGD (SEQ ID NO: 2)
SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHFL (SEQ ID NO: 3)
PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO: 4)
ANEAKVVLAADNVWGDNTGYQFLLDA (SEQ ID NO: 5)
ANEAKVVLAADNVWGDNTGYQFLLDA (SEQ ID NO: 5)
PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO: 4)
ADFTETFESSTHGEAPAEWTTIDA (SEQ ID NO: 6)
ADFTETFESSTHGEAPAEWTTIDA. (SEQ ID NO: 6)
It is presently preferred that the 9 proteins are PrtK48, PrtR45, PrtR44, PrtK39, PrtK44, PrtR27, PrtR17, PrtK15 and PrtR15 as described herein.
As the purified antigenic complex normally has enzymatic activity it is preferred in a number of uses the thiol endopeptidases are rendered inactive. This may be achieved in a number of ways, for example by oxidation or by mutation. It is presently preferred that the inactivation is by oxidation.
In yet another preferred embodiment of the present invention the multimeric protein complex is encoded by the DNA sequence shown in FIGS. 8B and 9B.
In a second aspect the present invention consists in a composition for use in eliciting an immune response directed against Porphyromonas gingivalis, the composition comprising an effective amount of the complex of the first aspect of the present invention and a suitable adjuvant and/or acceptable carrier.
In a third aspect the present invention consists in an antibody preparation comprising antibodies specifically directed against the complex of the first aspect of the present invention. The antibodies may be polyclonal antibodies or monoclonal antibodies.
In a fourth aspect the present invention consists in a method of treating a subject suffering from Porphyromonas gingivalis infection, the method comprising administering to the subject an amount of the antibody preparation of the third aspect of the present invention effective to at least partially neutralize the PrtR-PrtK complex of Porphyromonas gingivalis. 
As will be recognised by those skilled in the art the antibody preparation may be administered by any of a number of well known routes, however, it is presently preferred that the preparation is administered orally.
In a fifth aspect the present invention consists in a method of reducing the prospect of P. gingivalis infection in an individual and/or severity of disease, the method comprising administering to the individual an amount of the composition of the second aspect of the present invention effective to induce an immune response in the individual directed against P. gingivalis. 
In yet a further aspect the present invention consists in a recombinant host cell, the host cell being transformed with a DNA sequence(s) encoding PrtR-PrtK operatively linked to control sequences such that under appropriate conditions the host cell expresses PrtR-PrtK.
In another aspect, the present invention is directed to novel DNA sequences involving PrtR-PrtK constructs and vectors including plasmid DNA, and viral DNA such as human viruses, animal viruses, insect viruses, or bacteriophages which can be used to direct the expression of PrtR-PrtK protein in appropriate host cells from which the expressed protein may be purified. Another aspect of the present invention provides methods for molecular cloning of the genes encoding the PrtR-PrtK complex. The nucleic acid sequences of the present invention can be used in molecular diagnostic assays for P. gingivalis genetic material through nucleic acid hybridization, and including the synthesis of PrtR-PrtK sequence-specific oligonucleotides for use as primers and/or probes in amplifying, and detecting amplified, nucleic acids. Additionally, PrtR-PrtK complex can be used as an immunogen in prophylactic and/or therapeutic vaccine formulations against pathogenic strains of P. gingivalis, whether the immunogen is chemically synthesized, purified from P. gingivalis, or purified from a recombinant expression vector system. Alternatively, the genes encoding PrtR-PrtK may be incorporated into a bacterial or viral vaccine comprising recombinant bacteria or virus which is engineered to produce PrtR-PrtK by itself, or in combination with immunogenic epitopes of other pathogenic microorganisms. In addition, the genes encoding PrtR-PrtK operatively linked to one or more regulatory elements, can be introduced directly into humans to express the PrtR-PrtK to elicit a protective immune response. A vaccine can also be based upon a recombinant component of a mutated PrtR-PrtK incorporated into an appropriate vector and expressed in a suitable transformed host (eg. E. coli, Bacillus subtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells) containing the vector. The vaccine can be based on an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing an inactivated PrtR-PrtK is a commensal inhabitant of the oral cavity. Unlike whole P. gingivalis cells or other previously prepared antigens based on fimbriae or the capsule the PrtR-PrtK complex of the invention or component parts thereof are safe and effective antigens for the preparation of a vaccine for the prevention of P. gingivalis-associated periodontal disease. The invention therefore provides a range of recombinant products based on the PrtR-PrtK complex.
The invention also provides antibodies raised against the said PrtR-PrtK complex, herein called anti-PrtR-PrtK antibodies. The antibodies may be blended into oral compositions such as toothpaste, mouthwash, toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs.
In another aspect the invention provides a method of diagnosis for the presence of P. gingivalis characterised by the use of any one or a combination of an antibody, antigen or nucleic acid probe as hereinbefore defined comprising the application of known techniques including for example, enzyme linked immunosorbent assay.
The invention also provides diagnostic kits comprising antibodies, antigens and/or nucleic acid probes as hereinbefore defined.